Abstract Our work to date has highlighted new and unexpected roles for Ikaros and its associate, the chromatin remodeler Mi-2 in fate decisions made by hematopoietic stem cells and their early multipotent progeny. We now propose to further evaluate the roles of Ikaros and Mi-2 in cell fate restrictions in a newly identified lymphoid-primed multipotent progenitor (LMPP). The LMPP is the most proximal lymphoid progenitor to the hematopoietic stem cell (HSC) and a major branch-point of lymphoid and myeloid development. Our studies seek to delineate the Ikaros/Mi-2-based epigenetic machinery and its downstream genetic effectors that dictate the LMPP's choice for myeloid vs. lymphoid fate differentiation. Aim 1. To determine how LMPP fate determination is modulated by Ikaros and Mi-2. First, we examine the role of Ikaros as a repressor of self-renewal downstream of the HSC. Second, we test the role of Mi-2 as a negative regulator of lymphoid differentiation by studying its effects on lymphoid lineage priming and differentiation in the LMPP. Finally, the genetic interaction between Ikaros and Mi-2 and its effect on LMPP fate determination are elucidated. These studies seek to integrate previously described roles for Mi-2 and Ikaros as positive regulators of myeloid and lymphoid differentiation respectively, to determine the epistatic relationship between these two factors, and to ascertain their role in balancing the choice between innate vs. adaptive immunity. Aim 2. To obtain lineage-wide views of Ikaros and Mi-2 modes of action at the chromosome level. Here, we seek to obtain a genome-wide view of how these factors are deployed to generate progenitor-specific behaviors. This will reveal the stage-specific gene networks regulated by these factors and the role of Ikaros and Mi-2 in modulating transcriptional states during development. Interaction with other epigenetic regulators and their combined effects on chromatin structure during lymphoid development will be investigated. Aim 3. To identify the earliest effectors of lymphoid lineage restriction downstream of Ikaros and Mi-2. A functional analysis of Ikaros/Mi-2 targets expressed at the earliest point of lymphoid development is undertaken to discern how these may mediate the Ikaros and Mi-2 effects on lymphoid lineage restriction. A variety of genetic approaches are undertaken to manipulate their function both in vivo and in vitro. The effects of an initial set of nuclear and signaling molecules on the cascade of molecular and cellular events that sustains differentiation into the adaptive immune system are investigated.